Methods for studying human sirtuins with activity-based chemical probes

Zheng, Song Guo; Wohlfahrt, Jessica; Cohen, Ian; Cen, Yana

doi:10.1016/bs.mie.2019.11.004

Cited by 1 publication

(3 citation statements)

References 34 publications

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“…SIRT6 was a gift from Cheryl Arrowsmith (Addgene plasmid #28271). The proteins were expressed and purified according to previously published protocols [22,53]. The identity of the protein was confirmed by tryptic digestion followed by LC-MS/MS analysis performed at the Vermont Genetic Network (VGN) Proteomics Facility.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Recently, we developed a group of ABPs for human sirtuins (Figure 1, probes 1A-1C) [21,22]. They feature a thioacyllysine "warhead", a benzophenone photoaffinity group as well as a terminal alkyne bioorthogonal tag.…”

Section: Introductionmentioning

confidence: 99%

“…(A) Western blot showing increased acetylation level of H3K9 (H3K9Ac) in HEK293 cells treated with probe 2C; (B) Incubation with probe 2B or suramin increased CPS1Ac level in HEK293 cells.Encouraged by our initial cellular studies, we continued our effort on evaluating the dynamic enzyme activity change under different cellular conditions using ABPs in combination with imaging approach. Results from our lab and others have indicated that sirtuins have evolved to respond to the availability of NAD + , which is an essential currency of cellular metabolism and DNA repair, and convert this information to many different biological outputs[21,22,[41][42][43]. HEK293 cells overexpressing SIRT5 were subjected…”

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confidence: 99%

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Development of Second Generation Activity-Based Chemical Probes for Sirtuins

et al. 2020

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NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to low calorie conditions. They are thought to mediate the beneficial effects of calorie restriction to extend longevity and improve health profiles. Novel chemical probes are highly desired for a better understanding of sirtuin’s roles in various biological processes. We developed a group of remarkably simple activity-based chemical probes for the investigation of active sirtuin content in complex native proteomes. These probes harbor a thioacyllysine warhead, a diazirine photoaffinity tag, as well as a terminal alkyne bioorthogonal functional group. Compared to their benzophenone-containing counterparts, these new probes demonstrated improved labeling efficiency and sensitivity, shortened irradiation time, and reduced background signal. They were applied to the labeling of individual recombinant proteins, protein mixtures, and whole cell lysate. These cell permeable small molecule probes also enabled the cellular imaging of sirtuin activity change. Taken together, our study provides new chemical biology tools and future drug discovery strategies for perturbing the activity of different sirtuin isoforms.

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Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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